Scope of the Proposal This proposal seeks to characterize the basic biophysical mechanisms of nucleocytoplasmic transport to provide a firm understanding of how transport maintains, or through dysfunction fails to maintain in cells. Background/Intellectual Merit Nuclear pore complexes (NPCs) span the nuclear envelope and enable bidirectional transport between the cytoplasm and nucleus in eukaryotic cells. Small molecules (<40kDa) passively diffuse through the NPCs. However, translocation of large molecules (up to 50 MDa) is hindered by the phenylalanine-glycine (FG) repeats barrier inside the NPCs unless they are chaperoned by transport receptors. However, challenged by measuring the spatial structure of FG repeats and a series of transient interactions between the transport receptors and the FG repeats, the precise transport mechanism remains in dispute. To refine the transport mechanism, PI proposes an innovative expanded single molecule method to attack the challenges. Research Approach/Specific Aims The PI's lab develops a novel single-molecule approach, single-point edge-excitation sub-diffraction (SPEED) microscopy, to test nuclear transport models with a spatiotemporal resolution of 9 nm and 400 <s. SPEED microscopy enables us to capture transient interactions occurred in the NPC under real-time trafficking conditions that escape detection by previous single-molecule methods or electron microscopy. PI proposes studies with the following SPECIFIC AIMS: (1) Determine the spatial locations of transport pathways for small molecules;(2) Determine the spatial locations of transport pathways for large molecules;and (3) Determine the spatial distribution of the FG repeats at various trafficking conditions. Specific aim 1 &2 will examine the hypothesis that small molecules diffuse through a central channel, and that small and large molecules spatially separate their pathways. Specific aim 3 will examine whether the transport receptors repulse the FG-repeats filaments or dissolve into their meshwork, and whether the permeable barrier only anchors at the NPC center or spans the NPC. Significance/Broader Impacts Dysfunctions of the nuclear transport system are linked to numerous human diseases including leukemias, cancers, and primary biliary cirrhosis. The above proposed investigations will fundamentally advance our understanding of the nuclear transport mechanism and help distinguish among the current transport models. Advances expected from this work will directly impact our understanding and development of therapeutics for a number of human diseases. PUBLIC HEALTH RELEVANCE: Dysfunctions of the nuclear transport system are linked to numerous human diseases including leukemias, cancers, and primary biliary cirrhosis. Understanding of nuclear transport mechanism will directly impact our understanding and development of therapeutics for a number of human diseases. In this proposal, we employ novel single molecule approaches to further unravel the nuclear transport mechanism.